Elucidation of lipid nanoparticle surface structure in mRNA vaccines

Lipid nanoparticles (LNPs) have been used as a carrier for messenger RNA (mRNA) vaccines. Surface properties of LNPs are important to the stability and function of mRNA vaccines. Polyethylene-glycol (PEG) is a functional lipid at the surface of LNPs that improves colloidal stability, increases circulation time, and impacts cellular uptake. In this study, we explore in-depth lipid composition at the surface of mRNA-LNPs using high-field nuclear magnetic resonance (NMR) spectroscopy. Our results provide a unique surface lipid profile of intact LNPs identifying PEG chains and partial ionizable lipids are present with quantification capability. The surface PEG density is determined to reveal the brush-like conformation on the surface of mRNA-LNPs. Furthermore, we implement a diffusion NMR strategy for routine testing of formulated drug products during drug development. Comparative NMR analysis of different vaccine preparations and stability samples provides a global view of the mRNA-LNP surface structure for enhanced product knowledge.

Cholesterol 42.9 ± 0.1% Not detected N/A a qNMR method precision is within 5%.Please note NMR is performed as a non-GMP method for heightened characterization of various product quality attributes.b ALC-0159 abundance is < 2% according to COMIRNATY product insert.The larger deviation is due to the limited method precision at low concentration species.

Particle Size Distribution and Morphology: Dynamic Light Scattering (DLS), Asymmetric Flow Field Flow Fractionation (AF4), and Cryogenic Electron Microscopy (Cryo-EM)
The particle diameter and polydispersity index were measured by DLS using Malvern Zetasizer Ultra.The analysis was performed at a scattering angle of 90° at a temperature of 25 °C using samples diluted in 0.1xPBS.The size distribution profile (data not shown) represents a representative batch of LNPs with a mean diameter of 77 nm and a narrow size distribution (polydispersity index <1).
The size distribution and morphology of the COVID-19 lipid nanoparticle (LNP) were characterized by Asymmetric Flow Field Flow Fractionation coupled to a multi-angle light scattering detector equipped with in-line dynamic light scattering capability (AF4-MALS-DLS) (data not shown) and Cryo-EM.AF4-MALS-DLS is using ASTRA software from Wyatt Technologies, Inc.The in-line DLS is used to determine hydrodynamic radius (Rh) and the MALS is used to determine the Root Mean Squares radius (Rz).The shape factor (Rz/Rh) was used to determine the shape or morphology of the LNP.A solid sphere should have a shape factor of 0.77, a hollow sphere a shape factor of 1.0, and more elongated molecules greater than 1.The weight average shape factor of the LNP as 0.86, which indicates the spherical conformation of the particle.
To prepare for cryo-EM imaging, LNP samples (4uL) were applied in triplicate to a 200-mesh lacey formvar coated gold grids (Electron Microscopy Sciences) and were vitrified using a ThermoFisher Vitrobot Mark IV system (at 5C with 100% humidity).Samples were imaged using a Talos F200C transmission electron microscope equipped with Ceta 4kx4k camera and an electron accelerating voltage of 200 kV.Images were taken at a magnification of 36,000x and 45,000x (1 second exposure time).

LNP Density Measurement
Lipid nanoparticle (LNP) density was experimentally determined using the density match analytical ultracentrifugation (AUC) method as described in Henrickson et.al. 2 LNP samples were diluted in formulation buffer with varying amounts of formulation buffer made with D2O instead of water, resulting in varying buffer densities for each sample.A series of samples were made and SV-AUC experiments were performed.During analysis, only buffer viscosity was changed in the fitting algorithm, resulting in the movement of LNP species to lower s values.A plot of the movement of s values with buffer density gives a linear relationship (data not shown).By fitting a line to the data, and extrapolating to an s value of zero, the density of the particle was determined to be 1.0 g/mL.

Surface PEG Density Calculation
Detailed calculation protocol can be found in the previous work by Xu and co-worker. 3The PEG density [Γ] (PEG molecules/100 nm 2 ) was determined by surface PEG moles (MPEG, mole), total mass of nanoparticles (WNP, g), the density of nanoparticles (dNP, g/mL), and the particle diameter (D, nm) using Equation 1.The full surface coverage [Γ*] (unconstrained PEG molecules/ 100 nm 2 ) was determined by the molecular weight of the PEG chain (m, g/mol) using Equation 2 ) 2

Evaluation of the System Precision and the Method Precision for the NMR Method
A 1D 1 H diffusion experiment using Pulse Gradient Stimulated Echo (PGSTE)-bipolar gradients sequence was used for the LNP samples.The repeatability was evaluated by collecting triplicate measurements on a sample and preparing three different samples, respectively.LNP NMR spectra were recorded on a Bruker NEO 800 MHz spectrometer, equipped with a 5 mm protonoptimized triple resonance NMR inverse (TCI) cryoprobe at 25 °C (298 K).Spectra were processed and analyzed using MestreNova 14.1.

Figure S4. 1D 1 H
Figure S4.1D 1 H NMR Overlay of Intact mRNA-LNPs, ALC-0159 and ALC-0315 at Absolute Abundance Ratio Determined by Disrupted LNP Analysis.A, top spectrum: intact mRNA-LNPs in aqueous phosphate buffer; Bottom two spectra: individual lipids: ALC-0315 (aqueous, PBS), ALC-0159 (aqueous, PBS).Proton signals in the mRNA-LNP spectrum were labeled in green for ALC-0159, and in black for ALC-0315; overlapped signals from both lipids were labeled in italic.The detected surface protons were annotated in the molecular structures of ALC-0159 and ALC-0315.B, an expansion of 4.2-3.0ppm showing that in the intact LNP spectrum (black trace), the peak widths of ALC-0159-PEG (methylene and terminal methyl proton) signals are narrower than that of ALC-0315 peaks.In comparison of the LNP (black trace) and individual lipids in the aqueous environment (orange and green traces), the peak shapes are similar, because the individual lipids typically form micelles in the aqueous solution.